Note: Descriptions are shown in the official language in which they were submitted.
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USE OF PSILOCYBIN IN THE TREATMENT OF
NEUROLOGICAL BRAIN INJURY AND MIGRAINES
FIELD
The present invention relates to pharmaceutical compositions comprising
psilocybin and their use for the treatment of neurological brain injuries and
migraines.
BACKGROUND
Psilocybin (4-phosphoryloxy-N,N-dimethyltryptam me) is
a substituted
indolealkylamine and belongs to the group of hallucinogenic tryptamines.
Psilocybin is a
prodrug and undergoes dephosphorlyation to Psilocin in vivo. The chemical
formulas for
Psilocybin and Psilocin are:
Psitocybin Psilocin
0
HO¨P¨OH
0 OH
1.
= 3a 3 2
0 ,fl
,N,
7 7a N H3C CH3 N H3C CH3
Psilocybin was distributed worldwide under the name Indocybine (Sandoz) as a
short-acting and more compatible substance (than, for example, LSD) to support
is use
as a psychotherapeutic. Experimental and therapeutic use was extensive and
without
complications. (1)
Brain injury from a concussion is a complex condition which causes structural
damage and functional deficits from primary and secondary injury mechanisms,
respectively. (2) The primary injury mechanism is the result of the immediate
mechanical
disruption of brain tissue that occurs at the time of exposure to the external
force and
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includes, damage to blood vessels (hemorrhage), and axonal shearing, in which
the
axons of neurons are stretched and torn. The secondary injury mechanism
evolves over
minutes to months after the primary injury, and is the result of cascades of
metabolic,
cellular and molecular events that ultimately lead to brain cell death, tissue
damage and
atrophy in the injury boundary zone and subcortical regions.
Even in situations where there is minimal brain injury from a concussion,
cognitive
deficits, e.g., loss of memory, movement, sensation (e.g., vision or hearing)
or emotional
functioning (e.g., personality changes, depression) can result. Delayed
symptoms such
as irritability and other personality changes, sensitivity to light and noise,
sleep
disturbances, psychological adjustment problems such as depression and
disorders of
taste and smell may also result (2)
Typically for mild forms of brain injury, e.g concussion, bed rest, fluids,
and a mild
pain reliever such as acetaminophen (Tylenol) may be prescribed_ Ice may be
applied
to bumps to relieve pain and decrease swelling. Cuts are numbed with
medication such
as lidocaine, by injection or topical application. If needed, the wound
usually is closed
with skin staples, stitches (sutures), or, occasionally, a skin glue called
cyanoacrylate
(Dermabond).
However, there is no available treatment for the neurological brain damage and
the associated cognitive deficits experienced. (2)
Migraine is a common disabling primary headache disorder. Epidemiological
studies have documented its high prevalence and high socio-economic and
personal
impacts all over the world (Fendrich et al., Cephalalgia, 2007; 27:347-54; Le
et al., BMJ
Open, 2012; 2(4); Yong et al., J Headache Pain. 2012; 13:303-10; Yoon et al.,
J
Headache Pain. 2012; 13:215-23; Ertas et al., J Headache Pain. 2012; 13:147-
57).
Migraine is now ranked by the World Health Organization as number 19 among all
diseases world-wide causing disability.
Commonly starting at puberty, migraine most affects those aged between 20 and
50 years but can trouble much younger people, including children. The one-year
prevalence in adults is estimated to be 15%. In children and adolescents the
prevalence
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is approximately 5%. European and American studies have shown that 6-8% of men
and
15-18% of women experience migraine each year. The higher rates in women
everywhere (2-3 times those in men) are hormonally-driven. Prevalence declines
after 50
years of age (WHO Fact Sheet N 277, 2004; EMA CHMP Guideline, 2007).
There is no absolute cure for migraine since its pathophysiology has yet to be
fully
understood (Pietrobon & Striessnig, Nat Rev Neurosci. 2003; 4:386-98;
Cucchiara &
Detre, Med Hypotheses. 2008; 70:860-5). There are two medication strategies
for treating
migraine headaches. Treating the pain at the onset offers the best relief.
Over-the-counter
pain relievers such as acetaminophen, aspirin or other nonsteroidal anti-
inflammatory
drugs (NSAIDs) such as ibuprofen are commonly used (Pardutz & Schoenen,
Pharmaceuticals. 2010; 3:1966-1987). Prescription drugs such as triptans are
used for
headaches not relieved by over-the-counter medications. These are generally
not applied
to people who have high blood pressure or a heart disease. For those whose
headaches
are not adequately relieved with these medications, the second medication
strategy
involves medications prescribed prophylactically_ These are normally
prescribed to treat
other disorders but have been successful at reducing the frequency or severity
of
migraine headaches. Blood pressure medications such as beta-blockers or
calcium
channel blockers; antidepressant medications such as amitriptyline or
venlafaxine; and
anticonvulsant medications such as divalproex or topiramate (Hildreth et al.,
JAMA. 2009;
301:2608) have been used.
Any discussion of documents, acts, materials, devices, articles or the like
which
has been included in the present specification is not to be taken as an
admission that any
or all of these matters form part of the prior art base or were common general
knowledge
in the field relevant to the present invention as it existed before the
priority date of each
claim of this application.
SUMMARY OF THE INVENTION
The present disclosure, in one aspect, relates to a method for the treatment
of a
brain injury or a migraine in a mammal comprising administering a
therapeutically
effective amount of psilocybin or a pharmaceutically acceptable salt or
solvate thereof, to
a mammal in need thereof.
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The present disclosure, in another aspect, relates to a use of a
pharmaceutical
composition including psilocybin or a pharmaceutically acceptable salt or
solvate thereof,
together with one or more pharmaceutically acceptable carriers, diluents and
excipients
for the treatment of a brain injury or a migraine.
BRIEF DESCRIPTION OF THE DRAWINGS
For the purpose of illustrating the invention, the drawings show aspects of
one or
more embodiments of the invention. However, it should be understood that the
present
invention is not limited to the precise arrangements and instrumentalities
shown in the
drawings, wherein:
FIG. 1 is a schematic diagram of a timeline of an in vitro experiment, where
+AO
is with antioxidants and -A.O. is without antioxidants;
FIG. 2 is a schematic diagram of a timeline of an in vivo experiment;
FIG. 3A are photomicrographs representing MAP2 immunostaining;
FIG. 3B is a bar graph of MAP2 immunoreactivity;
FIG. 4A is a bar graph showing TBI significantly reduced duration stayed in
the target
zone;
FIG. 4B is a bar graph showing the average number of platform crossings;
FIG. 4C is a bar graph showing movement speed on PID3; and
FIG. 5A, FIG. 5B and FIG 5C are graphs showing high dose Psilocybin (PSI)
treatment significantly increased the time in the target area and the average
number of
platform crossings in TBI mice.
DETAILED DESCRIPTION
Throughout this specification the word "comprise", or variations such as
"comprises" or "comprising", will be understood to imply the inclusion of a
stated element,
integer or step, or group of elements, integers or steps, but not the
exclusion of any other
element, integer or step, or group of elements, integers or steps.
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For use in therapy a therapeutically effective amount of the psilocybin or
pharmaceutically acceptable salts or solvates thereof, may be presented as a
pharmaceutical composition. Thus, in a further embodiment the invention
provides a
pharmaceutical composition of psilocybin or pharmaceutically acceptable salts
or
solvates thereof in admixture with one or more pharmaceutically acceptable
carriers,
diluents, or excipients. The carrier(s), diluent(s) or excipient(s) must be
acceptable in the
sense of being compatible with the other ingredients of the formulation and
not deleterious
to the recipient thereof.
When applicable, the compositions of the present invention, including
psilocybin
may be in the form of and/or may be administered as a pharmaceutically
acceptable salt.
Typically, a pharmaceutically acceptable salt may be readily prepared by using
a
desired acid or base as appropriate. The salt may precipitate from solution
and be
collected by filtration or may be recovered by evaporation of the solvent.
Suitable addition salts are formed from acids which form non-toxic salts and
examples are hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate,
phosphate,
hydrogen phosphate, dihydrogen phosphate acetate, maleate, malate, fumarate,
lactate,
tartrate, citrate, formate, gluconate, succinate, pyruvate, oxalate,
oxaloacetate,
trifluoroacetate, saccharinate, benzoate, methanesulphonate, ethanesulphonate,
benzenesulphonate, p-toluenesulphonate and isethionate.
Suitable salts may also be formed from bases, forming salts including ammonium
salts, alkali metal salts such as those of sodium and potassium, alkaline
earth metal salts
such as those of calcium and magnesium.
Pharmaceutically acceptable salts may also be prepared from other salts,
including
other pharmaceutically acceptable salts, using conventional methods.
Those skilled in the art of organic or coordination chemistry will appreciate
that
many organic and coordination compounds can form complexes with solvents in
which
they are reacted or from which they are precipitated or crystallized. These
complexes are
known as "solvates". For example, a complex with water is known as a
"hydrate". Solvates
of psilocybin are within the scope of the present invention.
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Pharmaceutical compositions of the invention may be formulated for
administration
by any appropriate route, for example by the oral (including buccal or
sublingual).
Therefore, the pharmaceutical compositions of the invention may be formulated,
for
example, as tablets, capsules, powders, granules, lozenges, creams or liquid
preparations, such as oral solutions or suspensions. Such pharmaceutical
formulations
may be prepared by any method known in the art of pharmacy, for example by
bringing
into association the active ingredient with the carrier(s) or excipient(s).
Tablets and capsules for oral administration may be in unit dose presentation
form,
and may contain conventional excipients such as binding agents, for example
syrup,
acacia, gelatine, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for
example lactose,
sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting
lubricants, for
example magnesium stearate, talc, polyethylene glycol or silica;
disintegrants, for
example potato starch; or acceptable wetting agents such as sodium lauryl
sulphate. The
tablets may be coated according to methods well known in normal pharmaceutical
practice. Oral liquid preparations may be in the form of, for example, aqueous
or oily
suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a
dry product
for reconstitution with water or other suitable vehicle before use. Such
liquid preparations
may contain conventional additives, such as suspending agents, for example
sorbitol,
methyl cellulose, glucose syrup, gelatine, hydroxyethyl cellulose,
carboxymethyl
cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying
agents, for
example lecithin, sorbitan, monooleate, or acacia; non-aqueous vehicles (which
may
include edible oils), for example almond oil, oily esters such as glycerine,
propylene
glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-
hydroxybenzoate
or sorbic acid, and, if desired, conventional flavouring or colouring agents.
It should be understood that in addition to the ingredients particularly
mentioned
above, the formulations may include other agents conventional in the art
having regard
to the type of formulation in question.
The compositions of the present invention may be suitable for the treatment of
diseases in a human or animal patient. In one embodiment, the patient is a
mammal
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including a human, horse, dog, cat, sheep, cow, or primate. In one embodiment
the
patient is a human. In a further embodiment, the patient is not a human.
As used herein, the term "effective amount" means that amount of a drug or
pharmaceutical agent that will elicit the biological or medical response of a
tissue, system,
animal or human that is being sought, for instance, by a researcher or
clinician.
Furthermore, the term "therapeutically effective amount" means any amount
which, as
compared to a corresponding subject who has not received such amount, results
in
improved treatment, healing, prevention, or amelioration of a disease,
disorder, or side
effect, or a decrease in the rate of advancement of a disease or disorder. The
term also
includes within its scope amounts effective to enhance normal physiological
function.
In certain embodiments of the present invention, pharmaceutically acceptable
compositions of the present disclosure can be administered to humans and other
animals
at doses within the range of about 0.5 mL/day to about 3.0 mL/day and at a
concentration
within the range of about 0.5 mM to about 3.0 mM, particularly within the
range of about
0.5 mL/day to about 3.0 mL/day and at a concentration within the range of
about 0.5 mM
to about 1.0 mM, particularly within the range of about 0_5 mUday to about 3.0
mL/day
and at a concentration within the range of about 0.5 mM to about 1.5 mM,
particularly
within the range of about 0.5 mL/day to about 3.0 mL/day and at a
concentration within
the range of about 0.5 mM to about 2.0 mM, particularly within the range of
about 0.5
mL/day to about 3.0 mL/day and at a concentration within the range of about
0.5 mM to
about 2.5 mM, particularly within the range of about 0.5 mL/day to about 1.0
mL/day and
at a concentration within the range of about 0.5 mM to about 1.0 mM,
particularly within
the range of about 0.5 mL/day to about 1.5 mL/day at a concentration within
the range of
about 0.5 mM to about 1.0 mM, particularly within the range of about 0.5 mUday
to about
2.0 mL/day at a concentration within the range of about 0.5 mM to about 1.0
mM,
particularly within the range of about 0.5 mL/day to about 2.5 mL/day at a
concentration
within the range of about 0.5 mM to about 1_0 mM, particularly within the
range of about
0.5 mL/day to about 3.0 mL/day at a concentration within the range of about
0.5 mM to
about 1.0 mM, particularly within the range of about 0.5 mL/day to about 1.0
mL/day at a
concentration within the range of about 0.5 mM to about 1.5 mM, particularly
within the
range of about 0.5 mL/day to about 1.0 mL/day at a concentration within the
range of
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about 0.5 mM to about 1.5 mM, particularly within the range of about 0.5 mUday
to about
2.0 mL/day and at a concentration within the range of about 0.5 mM to about
1.5 mM,
particularly within the range of about 0.5 mUday to about 2.5 rnUday at a
concentration
within the range of about 0.5 mM to about 1_5 mM, particularly within the
range of about
0.5 mL/day to about 3.0 mL/day at a concentration within the range of about
0.5 mM to
about 1.5 mM, particularly within the range of about 0.5 mL/day to about 1.0
mL/day at
a concentration within the range of about 0.5 mM to about 2.0 mM, particularly
within the
range of about 0.5 mL/day to about 1.5 mL/day and at a concentration within
the range
of about 0.5 mM to about 2.0 mM, particularly within the range of about 0.5
mL/day to
about 2.0 mL/day at a concentration within the range of about 0.5 mM to about
2.0 mM,
particularly within the range of about 0.5 mUday to about 2.5 mL/day at a
concentration
within the range of about 0.5 mM to about 2_0 mM, particularly within the
range of about
0.5 mL/day to about 3.0 mL/day and at a concentration within the range of
about 0.5 mM
to about 2.0 mM, particularly within the range of about 0.5 mUday to about 1.0
mL/day
at a concentration within the range of about 0.5 mM to about 2.0 mM,
particularly within
the range of about 0.5 mL/day to about 1.5 mUday and at a concentration within
the range
of about 0.5 mM to about 2.0 mM, particularly within the range of about 0.5
mL/day to
about 2.0 mUday and at a concentration within the range of about 0.5 mM to
about 2.0
mM, particularly within the range of about 0.5 mL/day to about 2.5 mUday at a
concentration within the range of about 0.5 mM to about 2.0 mM, particularly
within the
range of about 0.5 mL/day to about 3.0 mL/day and at a concentration within
the range
of about 0.5 mM to about 2.0 mM, particularly within the range of about 0.5
mL/day to
about 1.0 mUday and at a concentration within the range of about 0.5 mM to
about 2.5
mM, particularly within the range of about 0.5 mL/day to about 1.5 mL/day at a
concentration within the range of about 0.5 mM to about 2.5 mM, particularly
within the
range of about 0.5 mL/day to about 2.0 mL/day and at a concentration within
the range
of about 0.5 mM to about 2.5 mM, particularly within the range of about 0.5
mL/day to
about 2.5 mUday and at a concentration within the range of about 0.5 mM to
about 2.5
mM, particularly within the range of about 0.5 mUday to about 3.0 mUday at a
concentration within the range of about 0.5 mM to about 2.5 mM, particularly a
dose of
0.5mUday at a 2 mM concentration, particularly a dose of 1.0 mL/day at a 2 mM
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concentration, particularly a dose of 1.2 mL/day at a 2 mM concentration,
particularly a
dose of 1.5 mL/day at a 2 mM concentration, particularly a dose of 2.0 mL/day
at a 2 mM
concentration, particularly a dose of 2.5 mL/day at a 2 mM concentration,
particularly a
dose of 3mL/day at a 2 mM concentration, particularly a dose within the range
of about
1.8 mL/day to about 2.2 mL/day at a concentration within the range of about
1.8 mM to
about 2.2 mM, particularly a dose within the range of about 1.9 mL/day to
about 2.1
mL/day at a concentration within the range of about 1.9 mM to about 2.1 mM,
particularly
within the range of about 0.5mUday to about 3.0 mL/day at a 2 mM
concentration,
particularly up to 3mL/day at a concentration up to 3.0 mM, particularly up to
3mL/day at
a 2 mM concentration, and this should provide a therapeutically effective
dose_ However,
the daily dose will necessarily be varied depending upon the host treated, the
particular
route of administration, and the severity of the illness being treated_
Accordingly, the
optimum dosage may be determined by the practitioner who is treating any
particular
patient. In another embodiment, the optimal dose may be higher.
As used herein the term "treatment" refers to defending against or inhibiting
a
symptom, treating a symptom, delaying the appearance of a symptom, reducing
the
severity of the development of a symptom, and/or reducing the number or type
of
symptoms suffered by an individual, as compared to not administering a
pharmaceutical
composition of the invention_ The term treatment encompasses the use in a
palliative
setting
Psilocybin is a strong agonist of the 5-HT2A receptor, as well as, a moderate
agonist at 5-HT1A and 5-HT2C.3 receptors. The 5-HT2A receptors are located
within the
thalamus and cortex of the brain. Activation of 5-HT2A receptors in the
thalamus, the area
of the brain responsible for sensory input, appears to decrease thalamic
activity, thus
leading to sensory alterations commonly referred to as hallucinations_ (3) Due
to this
alteration in sensory perception and serotonergic activity of psilocybin, much
of the
research for this agent has been focused on those mental health conditions
with
abnormalities in sensory perception, such as depressive disorders and anxiety
or anxiety-
related disorders. Note that these symptoms sometimes occur in concussions.
Psilocybin
has also been researched for use in substance use disorders. (3)
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However, 5-HT2A activity does not appear to account fully for psilocybin's
effect.
There is growing evidence that psilocybin might also be beneficial in treating
limiting brain
injury through its potential to contribute to brain complexity and plasticity.
(4) Therefore,
the present inventor postulates that psilocybin can reduce or eliminate the
common
cognitive and sensory deficit symptoms resulting from concussion through its 5-
HT2A
activity, as well as, help repair the limited brain injury resulting from a
concussion by its
contributing to brain complexity and plasticity, as well as, its capability to
stimulate
neurogenesis.
Scientists at the at University of California, Davis (UC Davis) have reported
that
psychedelic drugs (including psilocybin) promote neural plasticity and
development (4).
The UC Davis scientists treated cultures of cortical neurons with psychedelics
and
observed that the neurons developed and increased in complexity. They also saw
these
results in the brains of fly larvae and zebrafish, indicating that
psychedelics also have a
tangible effect in living organisms. In a separate experiment by the UC Davis
scientists,
psychedelics were found to significantly increase the number of dendritic
spines on
cortical neurons. Dendritic spines form synapses with other neurons and are a
major site
of molecular activity in the brain. Electrophysiological recordings found that
the frequency
and strength of neural currents were increased for many hours after the
psychedelic
compounds had been removed. Therefore, psychedelics may have the potential to
produce both structural and functional effects on neurons. (4)
The UC Davis scientists also attempted to determine the mechanism by which the
structural and functional effects occurred. It has been established that mTOR
regulates
neuronal development and plasticity and that its activity is disturbed in
neurodevelopmental and neurodegenerative diseases. (6) mTOR therefore was
blocked
and it was observed that the psychoplastogenic effects discussed above were
inhibited,
indicating that psychedelics may activate mTOR making this a potential
mechanism for
the neurogenesis activity. (4)
The UC Davis study builds on previous findings by the Beckley/Sant Pau
Research
Programme, which observed that components of the psychedelic brew
ayahuasca promoted growth and maturation of neurons. (7) The study also builds
upon
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reports in the literature from the 1950s, where it was found that LSD reversed
the sedating
effects of phenobarbital in cats (5, 8).
Based at least on the above results, the present inventor has a sound basis
for
predicting that neuron damage caused by a concussion can be mitigated through
psilocybin's promotion of structural and functional neural plasticity and
development for
treating traumatic brain injury (mild-TBI = concussion), more severe types of
TBI, stroke
and Alzheimer's.
Migraines are debilitating headaches caused by neurologic stimulation of blood
vessel
dilation in the brain.9 While they can be triggered by stress, anxiety,
fatigue or depression,
the root biological cause is unclear. Migraines carry a significant burden and
socioeconomic impact, having been found in 2013 to be the 6th leading cause of
years
lost to disability.10 Current therapies, including over-the-counter pain
relievers are
generally unsatisfactory in the relief of symptoms, and poor understanding of
the
biological cause has hampered the discovery of effective therapies for
migraines.9
Treatment of chronic or episodic migraines may also be approached with
preventive
drugs. Hemiplegic migraines, a type that is associated with weakness on one
side of the
body, are especially difficult to treat because of concerns about vessel spasm
and
stroke.ii A lack of good treatments for acute hem iplegic migraine makes
prevention using
safe daily administration of prophylactic compounds especially important.
Psilocybin is a strong activator of serotonin receptors, particularly 5-HT2,12
which is a
main mediator of serotonin signaling in the part of the brain known as the
hypothalamus.13
Irregularities in the neurotransmitter serotonin have long been known to be
associated
with chronic headaches involving brain vasculature, including migraines.14 In
fact,
serotonin deficiency has been discovered in families with genetic
predisposition to
hemiplegic migraines.is Such signaling in the thalamus and hypothalamus
regulate
sensory input and related disorders including anxiety and depression, which
psilocybin
has successfully been used to treat16 Low-dose psilocybin and other serotonin
receptor
activators can prevent another debilitating chronic headache disorder known as
cluster
headache (CH) and even induce remission.17 CH is characterized by abnormal
connectivity of the hypothalamus as has been demonstrated by fMRI brain
imaging.18
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Furthermore, direct stimulation of the hypothalamus effectively eliminated CH
in 10 of 16
patients and significantly reduced them in the other 6 in one study.19
Similarly, the
hypothalamus has been shown to be a mediator of chronic migraines as also
evidenced
by fMRI data.20 Thus, parallel brain activity irregularities involving the
hypothalamus are
likely to be at play in both CH and chronic migraines and are modulated by
serotonin
agonists, including psilocybin.
Psilocybin has proven safety in clinical doses that effectively mediate its
neurological
effects. Psilocybin has been extensively studied in clinical trials and
distributed worldwide
as a clinical therapy for anxiety and depression with a very safe toxicity
profile, even with
unsupervised administration.9 Based at least in part on the foregoing, and
together with
a mechanism of action on hypothalamic serotonin receptors, as observed in
cluster
headache, the present inventor has a sound basis for predicting that
psilocybin is an
effective drug for preventing or treating migraines, including those that are
difficult to treat
and require preventive therapies.
1. Summary of Experimental Study:
A study was designed to evaluate the therapeutic effect of Psilocybin in a
rodent
model of traumatic brain injury (TBI) and neuronal culture.
Psilocybin is a 5HT2a psychedelics, which increase BDNF expression and
neuritogenesis. These responses may improve neural repair after traumatic
brain injury.
In the current study, we conducted in vitro and in vivo experiments to
characterize the
therapeutic effect of Psilocybin in TBI animals.
(i) Neuroprotective effect of Psilocybin in primary cortical neuronal culture.
Cultured cells were treated with glutamate to simulate glutamate overflow in
brain
injury. Glutamate significantly reduced MAP-2 (microtubule-associated protein
2, a
neuronal marker) immunoreactivity. This reaction was not significantly altered
by
Psilocybin.
(ii) Neuroreparative effect of Psilocybin in a mouse model of
traumatic brain
injury.
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Adult mice were randomly assigned to 4 groups: (1) control, (2) TBI+veh, (3)
TBI+low
dose Psilocybin, and (4) high dose Psilocybin. TBI was delivered by a
Controlled
cortical impact (CCI). Vehicle (saline) or Psilocybin was given intranasally
injection
(i.n.) starting from 4 days after TBI for 5 days. Cognitive function was
examined by the
Morris water maze test after the injury. Animals were sacrificed for PCR or
protein
analysis after the behavior test. We found that Psilocybin at a high dose
significantly
improved cognitive function in the TBI mice.
These data support that Psilocybin, given after the injury, improves cognitive
function
in TBI mice.
2. Objective: This study was designed to evaluate the therapeutic effect of
Psilocybin in
a rodent model of traumatic brain injury and neuronal culture.
3. Experimental study details:
3-1: Methods and material:
A. Primary cultures of rat cortical neurons (PCN)
Primary cultures were prepared from embryonic (E14-15) cortex tissues obtained
from fetuses of timed pregnant Sprague-Dawley rats as we previously described
(1). The
olfactory bulbs, striatum, and hippocampus was removed aseptically; cortices
were
dissected. After removing the blood vessels and meninges, pooled cortices were
trypsinized (0.05 %; Invitrogen, Carlsbad, CA) for 20 min at room temperature.
After
rinsing off trypsin with pre-warmed Dulbecco's modified Eagle's medium
(Invitrogen),
cells were dissociated by trituration, counted, and plated into 96-well
(5.0x104/well) cell
culture plates precoated with poly-d-lysine (Sigma-Aldrich). The culture
plating medium
consisted of neurobasal medium supplemented with 2 % heat-inactivated fetal
bovine
serum (FBS), 0.5 mM L-glutamine, 0.025mM [-glutamate and 2 % B27 (Invitrogen).
Cultures were maintained at 37 C in a humidified atmosphere of 5 % CO2 and 95
% air.
The cultures were fed by exchanging 50 % of media with feed media (Neurobasal
medium, Invitrogen) with 0.5 mM L-glutamate and 2 % B27 with antioxidants
supplement
on days in vitro (DIV) 3 and 5. On DIV 7 and 10, cultures were fed with media
containing
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B27 supplement without antioxidants (Invitrogen). On DIV 10, cultures were
treated with
reagents. After 48hrs, cells were fixed at 4% paraformaldehyde for 1 hour at
room
temperature (please see the timeline in Fig 1).
B. Immunocytochemistry
Cells were fixed 48 hours after treatment of reagents using 4% P.F.A. After
removing
4% P.F.A. solution, cells were washed with phosphate-buffered saline (PBS).
Fixed cells
were treated with blocking solution [5% bovine serum albumin (B.S.A.) and 0.1%
Triton
X-100 (Sigma, St. Louis, MO, U.S.A.) in PBS] for 1 hour. The cells were
incubated for 1
day at 4 C with a mouse monoclonal antibody against MAP2 (1:500, Millipore,
Billerica,
MA, U.S.A.) and then rinsed three times with PBS. The bound primary antibody
was
visualized using Alexa Fluor 488 goat anti-mouse secondary (Invitrogen).
Images were
acquired using a camera DS-Qi2 (Nikon, Melville, NY) attached to a NIKON
ECLIPSE Ti2
(Nikon, Melville, NY). Data were analyzed using N.I.S. Elements AR 5.11
Software
(Nikon).
C. Animals:
Adult male CD1 mice were used for this study. Animals were randomly assigned
to 4
groups: (1) naïve (n=9), (2) TBI+veh (n=9), (3) TBI+low dose Psilocybin (n=7),
and (4)
high dose Psilocybin (n=7).
D. Controlled cortical impact (CCI) surgery and drug treatment
CCI: Mice were anesthetized with isoflurane and placed in a stereotaxic frame.
A
midline incision was made to expose the skull, and a 4 mm craniotomy was made
centered at -2 mm posterior to bregma and 0.5 mm lateral to midline over the
left
hemisphere. Mice were subjected to CCI at a 1.0 mm impact depth and a nominal
velocity
of 5 m/s. The dwell time was 500 ms, and the tip size was 2 mm. A computer-
controlled
pneumatically-driven piston from the CCI impactor device (TBI-0310 Impactor,
Precision
Systems and Instrumentation, Fairfax Station, VA) was used to impact the
brain. After the
impact, the head wound was sutured. Body temperature was maintained at 37 C
using a
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temperature-controlled incubator. Control animals received sham surgery,
including
craniotomy without cortical impact.
Intranasal drug delivery: Animals were anesthetized with isoflurane each day
and
were placed in a supine position. Psilocybin (high dose: 50 pM in 20 pl
saline; low dose:
50 pM in 10 pl saline, Cayman Chemical, Michigan, U.S.A.) or saline (20 pl)
was delivered
into nostrils of each mouse per day from day 4 to day 8 (total 5 days) after
CU. No animal
died during surgery or during post-TBI drug treatment.
E. Morris water maze test:
Cognitive function was assessed using the Morris water maze (MWM) test. A
circular
tank 200 cm in diameter, 45 cm in height, was filled with water maintained
between 25-
28 C_ All animals received 5-day training prior to the T B I (see timeline,
Fig 2)_ In the
training period, the water level was lowered in the tank so that the surface
of the platform
(8 cm in diameter) was 1.5 cm above the water level. The animal was allowed to
swim for
60 seconds in the pool to locate the platform. After the platform was located,
the animal
was allowed to remain on the platform for 30 seconds. If the animal did not
locate the
platform within 60 seconds, it was gently guided to it by the experimenter and
allowed to
remain for 30 seconds.
On post-injury days (PID) 3, 10, 14, and 21, animals were evaluated in 60 s
probe
trials without the escape platform. The swim path of a mouse during each trial
will be
recorded by a video camera connected to a tracking system. Latency time and
the length
of swim path were recorded. The locomotor activity of the mice was analyzed
using an
average swim speed. The spatial memory for the platform location during probe
trials was
evaluated by the analysis of the dwelling duration (in sec) and the number of
times the
animal crossed the platform zone, defined as 3x the diameter of the platform
(i_e_, 24 cm
diameter, or an additional 8 cm radius beyond the platform perimeter). All
parameters
were automatically recorded and analyzed by video tracking software (Etho
vision XT 8_5,
Noldus, Leesburg, VA, U.S.A.).
G. Statistical Analysis
Data were presented as mean s.e.m. One or two¨way ANOVA and post-hoc Fisher
tests were used for statistical comparisons, with a significance level of
p<0.05.
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3-2: Results
A. Psilocybin (PSI) did not significantly protect against glutamate
neurotoxicity in
primary cortical neuronal culture
Glutamate (Glu) -mediated neuronal loss was examined by MAP-2 immunostaining.
Typical photomicrographs were shown in Fig 3A. The MAP2 immunoreactivity (MAP2-
ir)
was quantified and averaged to the mean of vehicle control group (Fig 3B). Glu
(100 pM)
significantly reduced MAP2-ir (Fig 3B1, Glu vs. veh, p<0.001, F3,19 =29.361,
one-way
ANOVA+ post hoc Fisher test). There is a non-significant trend that Psilocybin
may
partially antagonize GLU-mediated neurodegenerative response (p=0.062, Glu vs.
Glu+100 nM PSI.; p=0.061, Glu vs. Glu+1pM PSI; Fig 3B).
With referene to FIG.3, Psilocybin (Psi) did not antagonize glutamate (Glu) -
mediated
neuronal loss in primary cortical neuronal culture (PCN). FIG. 3 (A)
Representing MAP2
immunostaining. FIG. 3(B) The MAP2-ir was averaged to the mean of vehicle
control
group. Glu treatment significantly reduced MAP2-ir (p<0.001). Glu-mediated
MAP2-ir
reduction was not significantly antagonized by Psilocybin (p>0.05, one-way
ANOVA +
post-hoc Fisher test). Data are represented as mean +1- S.E.M., n=5-6 per each
group.
B. Water maze test:
A total of 32 mice were used for this study. Adult mice were randomly assigned
to 4
groups: (1) naïve (n=9), (2) TBI+veh (n=9), (3) TBI+low dose Psilocybin (n=7),
and (4)
high dose Psilocybin (n=7).
On post-injury day 3 (PID3) before drug treatment, animals received a single
60-second
water maze test with the platform removed. As seen in Fig 4, TBI significantly
reduced
(A) duration stayed in the target zone, (b) the average number of platform
crossings, and
(c) movement speed. These data can also be found in Table 1-3.
Table 1: Time in the target area in MWM test on PID3 (before drug treatment)
and PIDs
10, 14,21 (after drug treatment)
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Time in the target area (sec)
n= Day 3 Day 10 Day 14 Day 21
Naïve 9 5.33 0.49 5.97 0.54 3.75 0.55 4.40 0.82
CCI 9 2.38 0.60 3.21 0.30 2.68 0.48 2.74 0.41
Psi-H 7 2.89 0.57 312 0.26 3.45 0.18 3.88 0.18
Psi-L 7 3.21 0.84 287 045 2.82 0.45 2.51 0.41
Table 2: Average number of plafform crossings in MWM test on PID3 (before drug
treatment) and PIDs 10, 14,21 (after drug treatment)
No. of crossing
n= Day 3 Day 10 Day 14 Day 21
Naïve 9 9.00 0.83 8_33 0.78 5.44 0.78 6.11 0/7
CCI 9 4_00 0_91 4_89 0_59 3_89 0_51 5_00 0.47
Psi-H 7 4.43 0.69 6.00 0.85 6.00 0.44 6.00 0.66
Psi-L 7 3.57 0.90 4.14 0.51 4.43 0.61 3.29 0.57
Table 3: Movement speed in MWM test on PID 3 (before drug treatment) and PIDs
10,
14, 21 (after drug treatment)
Velocity (cm/sec)
n= Day 3 Day 10 Day 14 Day 21
Naïve 9 29.47 0.81 28.59 0.55 2T19 0.84 2T23 1.29
CCI 9 27.00 1.07 27.23 0.96 27.08 1.1025.93 1.17
Psi-H 7 23.27 1.40 25.00 1.57 26.38 1.22 27.06
1.53
Psi-L 7 26.35 1.44 25/9 1.39 26.18 0/6 26.66 0/6
FIG. 4A is a bar graph showing TBI significantly reduced duration stayed in
the target
zone, FIG. 4B is a bar graph showing the average number of platform crossings,
and FIG.
4C is a bar graph showing movement speed on PID3. * denotes two-tailed
student's t-
test.
After receiving drug or vehicle treatment from day 4 to day 8, animals were re-
evaluated in 60 s probe trials without the escape platform on PID 10, 14, and
21.
As seen in Fig 5, high dose Psilocybin treatment significantly increased the
time in the
target area and the average number of platform crossings in TBI mice (p values
is shown
in Table 4-5).
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Table 4. Significant improvement in Water maze test after a high dose
Psilocybin
treatment.
Time in the target No. of
area (sec) crossing
TBI TBI
naive P<0.001 P<0.001
Psi-
P=0.045 P=0.011
Psi-L P=0.712 P=0.237
All data were compared to the animals receive TBI and vehicle.
P-value was calculated by a two way ANOVA +posthoc Fisher test.
Table 5. Significant improvement in Water maze test after drug treatment.
Time in the target No. of
area (sec) crossing
vs naive naive
T.B.I. P<0.001 P<0.001
Psi-
P=0.011 P=0.245
Psi-L P<0.001 P<0.001
All data were compared to the naïve animals.
P-value was calculated by a two way ANOVA +posthoc Fisher test.
The movement velocity was not altered amongst all groups. (see mean and S.E.M.
in
Table 1-3). CCI or Psilocybin did not significantly alter body weight (Table
6).
Table 6. CCI or Psilocybin treatment did not significantly alter body weight
body
n=
weight (g)
Naive 9 35.67 0.53
CCI 9 36.78 0.70
Psi-H 7 34.7 0.87
Psi-L 7 35.71 0.68
The bodyweight of animals was measured on PID21
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FIG. 5A, 5B and 5C are graphs showing high dose Psilocybin (PSI) treatment
significantly increased the time in the target area and the average number of
platform
crossings in TBI mice. The velocity of movement was not altered. See also the
p-value in
Tables 4 and 5.
4. Discussion:
In this study, the potential therapeutic effect of Psilocybin in neuronal
culture and
experimental animals was characterized. It was first demonstrated that
Psilocybin did not
significantly alter glutamate-mediated loss of MAP2-ir in primary cortical
neurons.
Using a TBI mouse model, it was found that post-treatment with Psilocybin
improved
cognitive function as seen in the Morris water maze test. The cognitive
improvement by
high dose Psilocybin may be attributed to its reparative action as no
significant protection
was found in neuronal culture.
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